Although a variety of methods have been described for preparing conjugates of immunoglobulins and detectable labels, such as antibody-enzyme conjugates, such methods have resulted in random conjugation at many sites leading to heterogeneity and lack of reproducibility in the conjugates.
Site-specific carbohydrate labeling of the Fc region of antibodies with enzymes has also been described in O'Shannessy, et al., "Labeling of the Oligosaccharide Moieties of Immunoglobulins", Journal of Immunological Methods, Vol. 99 (1987) (pages 153-161). However, conjugates prepared according to such method result in distances between the enzyme and the antibody which are intrinsically short, leading to distorted, conformationally strained antibodies and/or enzymes.
European Patent Application No. 32,863 describes conjugation methods and conjugation agents for linking compounds containing carbohydrate moieties or carboxyl groups to compounds containing thiol moieties or electron-deficient moieties. In particular, the coupling of immunoglobulins at the Fc region to labels, such as enzymes, is described. However, such methods and linking reagents used thereby provide reaction conditions whereby there is essentially no discrimination between the disulfides on the Fc region and the complementarity defined region of the immunoglobulin. This lack of discrimination results in alteration of the complementarity defined region by reduction of disulfide bonds therein to thereby affect the binding sites on the immunoglobulin.
Accordingly, there is a need for a technique to prepare site-specific conjugates which avoids reductive cleavage of the disulfide bonds in, and deterioration of, the complementarity defined region of the antibody.